WO2005023177A2 - Anticorps monoclonaux humains diriges contre l'antigene protecteur de bacillus anthracis - Google Patents

Anticorps monoclonaux humains diriges contre l'antigene protecteur de bacillus anthracis Download PDF

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WO2005023177A2
WO2005023177A2 PCT/US2004/016213 US2004016213W WO2005023177A2 WO 2005023177 A2 WO2005023177 A2 WO 2005023177A2 US 2004016213 W US2004016213 W US 2004016213W WO 2005023177 A2 WO2005023177 A2 WO 2005023177A2
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antibody
human
anthrax
antibodies
protective antigen
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PCT/US2004/016213
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WO2005023177A3 (fr
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Tibor Keler
Diann Blanset
Laura A. Vitale
Israel Lowy
Mohan Srinivasan
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Medarex, Inc.
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Priority to CA2526398A priority Critical patent/CA2526398C/fr
Priority to MXPA05012571A priority patent/MXPA05012571A/es
Priority to JP2006514938A priority patent/JP4999158B2/ja
Priority to AU2004270103A priority patent/AU2004270103B2/en
Priority to EP04809410A priority patent/EP1633785B1/fr
Publication of WO2005023177A2 publication Critical patent/WO2005023177A2/fr
Publication of WO2005023177A3 publication Critical patent/WO2005023177A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1278—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Bacillus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1009—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against material from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • Anthrax (Bacillus anthracis) is primarily a disease of domesticated and wild animals, particularly herbivorous animals, such as cattle, sheep, horses, mules, and goats. Although natural anthrax infection in humans is rare (risk of infection through contact with diseased animals is about 1/100,000), it poses a very real threat from bioterrorism. The bacteria form hardy spores which are heat resistant and can survive for decades under crude conditions. Although cutaneous anthrax is more readily treatable, inhalation anthrax typically results in an abrupt catastrophic illness having a mortality rate of greater than 80% in 2-4 days.
  • the capsule is more important during the establishment of the infection than in the terminal phases of the disease, which are mediated by the anthrax toxin.
  • the toxin which is responsible for the disease etiology, is composed of protective antigen (PA), lethal factor (LF) and edema factor (EF).
  • PA protective antigen
  • LF lethal factor
  • EF edema factor
  • the EF is a calcium-calmodulm-dependent adenylate cyclase believed to cause the edema associated with anthrax infection and to prevent immune cells from ingesting and degrading the bacteria.
  • the LF is a cell-type specific metalloprotease that cleaves mitogen-activated protein kinase-kinases and several peptide hormones.
  • LF is the major virulence factor associated with anthrax toxicity and is responsible for systemic shock and death. None of the toxin components are pathogenic alone, and EF and LF require PA to exert their toxic effects inside a host cell.
  • an 83 kDa protective antigen PA83 protein secreted from rapidly growing B. anthracis binds to the host's cell surface via the anthrax toxin receptor (ATR) (Bradley et al, 2001, Nature 414:225-229).
  • ATR anthrax toxin receptor
  • the pore formed by PA63 permits transport of LF and EF into the cytoplasm where they can elicit their respective toxicities.
  • Soluble ATR sATR introduced into media containing ATR-bearing cells (e.g., macrophages), causes PA to bind to the sATR instead of the receptor on the cell surface (Bradley et al., 2003, Biochem. Pharmacol. 65: 309-314) suggesting that ATR may be useful in the design of anthrax treatments.
  • DNIs dominant negative inhibitors
  • Anthrax vaccine which contains PA as the primary immunogenic component, may confer protection against the disease.
  • the immunization schedule e.g., six initial doses followed by yearly boosters
  • the immunization schedule does not generate strong immunological memory.
  • a lack of standardization of the level of antigen results in a high degree of variability in efficacy on a lot-lot basis.
  • the vaccine since the vaccine is a cell-free culture media filtrate, which contains several cellular components, it may contribute to a high incidence of local and systemic reactions.
  • Monoclonal antibodies have advantages over antibiotics, but also which can be used to augment antibody efficacy.
  • antibiotics will not neutralize the preformed and released toxin that is causing the pathology, whereas antibodies can neutralize additional toxin before it can contribute further to the inflammatory cascade.
  • prophylactic treatment the duration of required antibiotic prophylaxis is 60 days, which can be difficult to follow. Furthermore, this time span may be covered by a single injection of antibody.
  • antibiotics can interfere with the development of a protective immune response so that there is no protection afforded after dosing is terminated. Accordingly, there is a need for improved therapies for treating anthrax infection, particularly antibodies against B. anthracis protective antigen, which will be well tolerated by the immune system and capable of prophylactic, post-exposure prophylactic, and therapeutic uses.
  • the present invention provides isolated human monoclonal antibodies, which bind to the protective antigen of Bacillus anthracis (anthrax) thereby inhibiting its biological activity, as well as derivatives (e.g., immunoconjugates, bispecific molecules and single chain fragments (ScFv) and other therapeutic compositions containing such antibodies, alone or in combination with additional therapeutic agents. Also provided are methods and compositions for treating and preventing anthrax infection using the antibodies of the invention.
  • Antibodies of the present invention provide an improved means for treating and preventing anthrax infection attributable in part to the antibodies' ability to effectively neutralize protective antigen activity and to the antibodies' fully human composition, which makes them significantly less immunogenic and more therapeutically effective and useful when administered to human patients than other protective antigen antibodies previously generated (e.g., murine and humanized antibodies).
  • the invention provides fully human antibodies that ⁇ ⁇ 7 1 bind to Bacillus anthracis protective antigen with an apparent affinity of at least 10 M " and neutralize a Bacillus anthracis toxin at an ED 50 of 5 ⁇ g/ml or less in a toxin neturalization assay.
  • the invention provides neutralizing human monoclonal antibodies against anthrax protective antigen, which have one or more of the following characteristics: (1) requires binding to Fc receptor for neutralization activity; (2) exhibits higher affinity for PA 63 over PA 83; (3) exhibits one or more characteristics selected from: (a) does not bind PA 83 at 1 ⁇ g ml, OD 40 5nm 0.2 or less according to standard ELISA, (b) does not block anthrax lethal factor or edema factor from binding protective antigen in a competitive binding assay; (4) does not compete with an anti-PA antibody selected from murine 1 G3, murine 2D5 and murine 14B7 in binding to PA 63.
  • the antibody has a human heavy chain variable region and a human light chain variable region where the sequences of the human heavy chain variable region and the human light chain variable region respectively comprise the sequences selected from SEQ ID NOs: 2 and 4, SEQ ID NOs:
  • the antibody has a human heavy chain variable region containing FR1, CDRl, FR2, CDR2, FR3, CDR3 and FR4 sequences and a human light chain variable region comprising FR1, CDRl, FR2, CDR2, FR3, CDR3 and FR4 sequences, where the human heavy chain variable region CDR3 sequence is selected from SEQ ID NOs: 20, 38, 50 and 62, and conservative modifications thereof; and the human light chain variable region CDR3 sequence is selected from SEQ ID NOs: 26, 32, 44, 56 and 68, and conservative modifications thereof.
  • particular human antibodies of the invention include those which comprise a CDR domain having a human heavy and light chain CDRl region, a human heavy and light chain CDR2 region, and a human heavy and light chain CDR3 region, which comprise an amino acid sequence at least 80% homologous, preferably 85% homologous, more preferably 90%, 95%, 98%, or 99% homologous to the amino acid sequences of the CDRl, CDR2, and CDR3 regions shown in Figures 1-9.
  • the antibody has a human heavy chain variable region and a human light chain variable region, where the human heavy chain variable region contains an amino acid sequence selected from SEQ ID NOs: 2, 8, 12 and 16, and sequences that are at least 80% homologous to SEQ ID NOs: 2, 8, 12 and 16, and the human light chain variable region contains an amino acid sequence selected from SEQ ID NOs: 4, 6, 10, 14 and 18, and sequences that are at least 80% homologous to SEQ ID NOs: 4, 6, 10, 14 and 18.
  • a particular therapeutic antibody of the present invention includes human monoclonal antibody (HuMab) 5E8 and functionally equivalent antibodies which (a) are encoded by human heavy chain and human light chain nucleic acids comprising nucleotide sequences in their variable regions as set forth in SEQ ID NO:l and SEQ ID NO:3, respectively, and conservative sequence modifications thereof, or (b) include heavy chain and light chain variable regions which comprise the amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4, respectively, and conservative sequence modifications thereof, hi another embodiment, the light chain variable region of HuMab 5E8 comprises a nucleotide sequence and amino acid sequence as set forth in SEQ ID NOs: 5 and 6, respectively, including conservative modifications thereof.
  • Another particular therapeutic antibody of the present invention includes human monoclonal antibody 2D5 and functionally equivalent antibodies which (a) are encoded by human heavy chain and human light chain nucleic acids comprising nucleotide sequences in their variable regions as set forth in SEQ ID NO: 7 and SEQ ID NO:9, respectively, and conservative sequence modifications thereof, or (b) include heavy chain and light chain variable regions which comprise the amino acid sequences shown in SEQ ID NO:8 and SEQ ID NO: 10, respectively, and conservative sequence modifications thereof.
  • Yet another particular therapeutic antibody of the present invention includes human monoclonal antibody 2H4 and functionally equivalent antibodies which (a) are encoded by human heavy chain and human light chain nucleic acids comprising nucleotide sequences in their variable regions as set forth in SEQ ID NO:l 1 and SEQ ID NO:13, respectively, and conservative sequence modifications thereof, or (b) include heavy chain and light chain variable regions which comprise the amino acid sequences shown in SEQ ID NO: 12 and SEQ ID NO: 14, respectively, and conservative sequence modifications thereof.
  • Still another particular therapeutic antibody of the present invention includes human monoclonal antibody 5D5 and functionally equivalent antibodies which (a) are encoded by human heavy chain and human light chain nucleic acids comprising nucleotide sequences in their variable regions as set forth in SEQ ID NO: 15 and SEQ ID NO: 17, respectively, and conservative sequence modifications thereof, or (b) include heavy chain and light chain variable regions which comprise the amino acid sequences shown in SEQ ID NO: 16 and SEQ ID NO: 18, respectively, and conservative sequence modifications thereof.
  • the invention further encompasses antibodies that bind to an epitope on anthrax protective antigen defined by antibody 5E8, 2D5, 2H4 or 5D5, and/or which compete for binding to protective antigen with antibody 5E8, 2D5, 2H4 or 5D5, or which have other functional binding characteristics exhibited by antibody 5E8, 2D5, 2H4 or 5D5.
  • Such antibodies include those which specifically bind to protective antigen (e.g., no cross-reactivity with cell-surface antigens) and exhibit toxin neutralizing activity.
  • the invention provides nucleic acid molecules encoding human anti-PA antibodies and portions thereof (e.g., variable regions thereof), as well as recombinant expression vectors which include the nucleic acids of the invention, and host cells transfected with such vectors. Methods of producing the antibodies by culturing these host cells are also encompassed by the invention.
  • the invention provides a transgenic non-human animal having a genome comprising a human heavy chain transgene or transchromosome and a human light chain transgene or transchromosome, which express human monoclonal antibodies that bind to protective antigen.
  • the transgenic non-human animal is a transgenic mouse (also referred to herein as a "HuMAb mouse®").
  • the invention provides isolated B-cells from a transgenic non-human animal as described, e.g., a transgenic mouse, which expresses human anti-PA antibodies.
  • the isolated B-cells can then be immortalized by fusion to an immortalized cell to provide a source (e.g., a hybridoma) of human anti-PA antibodies.
  • a source e.g., a hybridoma
  • hybridomas i.e., which produce human anti-protective antigen antibodies
  • human antibodies of the invention can be obtained directly from hybridomas which express the antibody, or can be cloned and recombinantly expressed in a host cell (e.g., a CHO cell or a lymphocytic cell). Accordingly, in another aspect, the present invention provides methods for producing human monoclonal antibodies which bind to anthrax protective antigen by immunizing with B.
  • anthracis protective antigen or a cell expressing B. anthracis protective antigen, a transgenic non-human animal having a genome containing a human heavy chain transgene and a human light chain transgene, such that antibodies are produced by B cells of the animal, isolating the B cells, and fusing the B cells with myeloma cells to form immortal hybridoma cells that secrete the antibody.
  • the method includes immunizing a HuMAb Mouse® with a purified or enriched preparation of anthrax protective antigen and/or cells expressing anthrax protective antigen.
  • human anti-PA antibodies of the invention are derivatized, linked to, or co-expressed with another functional molecule, e.g., another peptide or protein (e.g., a Fab' fragment).
  • another functional molecule e.g., another peptide or protein (e.g., a Fab' fragment).
  • an antibody or antigen-binding portion of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody to produce a bispecific or a multispecific antibody.
  • the present invention encompasses a large variety of antibody conjugates, bispecific and multispecific molecules, and fusion proteins, all of which bind to anthrax protective antigen, and can be used to target the anthrax protective antigen to particular cells.
  • the present invention provides a method for detecting in vitro or in vivo the presence of anthrax protective antigen in a sample, e.g., for diagnosing anthrax infection. In one embodiment, this is achieved by contacting a sample to be tested, optionally along with a control sample, with a human monoclonal antibody of the invention (or an antigen-binding portion thereof) under conditions that allow for formation of a complex between the antibody and protective antigen followed by detection of the complex formed (e.g., using an ELISA).
  • a control sample along with the test sample complex is detected in both samples and any statistically significant difference in the formation of complexes between the samples is indicative the presence of protective antigen in the test sample.
  • the present invention provides methods of screening for an antibody against anthrax protective antigen by the sequential steps of selecting one or more antibodies which neutralizes anthrax toxin in a toxin neutralization assay, followed by selecting an antibody which has an ED 50 of 0.1 g/ml or less, where an antigen- antibody binding affinity assay is not used as a selection criterion prior to either step.
  • the present mvention provides therapeutic and diagnostic compositions comprising one or more human anti-PA antibodies together with a carrier.
  • the composition further includes one or more additional therapeutic agents, such as a protective antigen vaccine or a second antibody against anthrax bacteria, spores, protective antigen, lethal factor or edema factor, or a Fab, F(ab') 2 , Fv or single chain Fv fragment of the second antibody.
  • additional therapeutic agents such as a protective antigen vaccine or a second antibody against anthrax bacteria, spores, protective antigen, lethal factor or edema factor, or a Fab, F(ab') 2 , Fv or single chain Fv fragment of the second antibody.
  • the invention provides methods of in vivo treatment and prevention of anthrax by administering human antibodies of the present mvention to patients (e.g., human subjects) at therapeutically effective dosages using any suitable route of administration known in the art for antibody-based clinical products, e.g., SC injection and intravenous.
  • the invention provides methods for treating or preventing anthrax in a patient in need of such treatment by administering a neutralizing antibody against anthrax protective antigen, having the characteristics that (1) it requires binding to Fc receptor for neutralization activity, (2) exhibits higher affinity for PA 63 over PA 83, (3) exhibits one or more of (a) does not bind PA 83 at 1 ju.g/ml, OD 405nrn 0.2 or less according to standard ELISA, (b) does not block anthrax lethal factor or edema factor from binding protective antigen in a competitive binding assay (e.g., see Little, 1996), and (4) does not compete with an anti-PA antibody selected from murine 1G3, murine 2D5 and murine 14B7 in binding to PA 63, in a dosage from 0.1 mg/kg to 100 mg/kg.
  • a neutralizing antibody against anthrax protective antigen having the characteristics that (1) it requires binding to Fc receptor for neutralization activity, (2) exhibits higher affinity for PA 63 over PA 83, (3)
  • patients infected with anthrax and exhibiting signs and/or symptoms of anthrax disease can be treated with an antibody of the invention.
  • human antibodies of the invention are co- administered with one or more additional therapeutic agents, e.g., an antibiotic, a protective antigen vaccine or antibodies against anthrax bacteria, spores, protective antigen, lethal factor or edema factor.
  • additional agents can be co-administered simultaneously with administration of an antibody of the invention (e.g., in a single composition or separately) or administered before or after administration of the anti-PA antibody.
  • Figure 1 shows the nucleotide sequence (SEQ ID NO: 1) and corresponding amino acid sequence (SEQ ID NO: 2) of the V H -region from HuMab 5E8. CDR regions are indicated.
  • Figure 2 shows the nucleotide sequence (SEQ ID NO: 3) and corresponding amino acid sequence (SEQ ID NO: 4) of the V L -region from HuMab 5E8 (major). CDR regions are indicated.
  • Figure 3 shows the nucleotide sequence (SEQ ID NO: 5) and corresponding amino acid sequence (SEQ ID NO: 6) of the V H -region from HuMab 5E8 (minor). CDR regions are indicated.
  • Figure 4 shows the nucleotide sequence (SEQ ID NO: 9) and corresponding amino acid sequence (SEQ ID NO: 10) of the V H -region from HuMab 2D5. CDR regions are indicated.
  • Figure 5 shows the nucleotide sequence (SEQ ID NO: 11) and corresponding amino acid sequence (SEQ ID NO: 12) of the V L -region from HuMab 2D5. CDR regions are indicated.
  • Figure 6 shows the nucleotide sequence (SEQ ID NO: 13) and corresponding amino acid sequence (SEQ ID NO: 14) of the Vu-region from HuMab 2H4. CDR regions are indicated.
  • Figure 7 shows the nucleotide sequence (SEQ ID NO: 15) and corresponding amino acid sequence (SEQ ID NO: 16) of the V L -region from HuMab 2H4. CDR regions are indicated.
  • Figure 8 shows the nucleotide sequence (SEQ ID NO: 17) and corresponding amino acid sequence (SEQ ID NO: 18) of the VH-region from HuMab 5D5-2E10. CDR regions are indicated.
  • Figure 9 shows the nucleotide sequence (SEQ ID NO: 19) and corresponding amino acid sequence (SEQ ID NO: 20) of the VL-region from HuMab 5D5-2E10. CDR regions are indicated.
  • Figure 10 is a graph showing neutralization of B. anthracis lethal toxin by four different human anti-PA antibodies.
  • Figure 11 is a graph showing anti-PA antibodies binding by ELISA to full-length protective anitgen (83kD; white bars) and cleaved protective antigen (63kD; hatched bars).
  • Figure 12 is a graph showing a kinetic analysis of neutralization of B. anthracis lethal toxin by HuMAb 5E8 and murine mAb 14B7.
  • Figure 13 is a graph showing comparison of neutralization of B. anthracis lethal toxin using the F(ab') 2 fragment of HuMAb 5E8 antibody and the intact 5E8 mAb in the absence or presence of anti-mouse FcRII/i ⁇ monoclonal antibody 2.4G2.
  • Figure 14 is a graph showing comparison of binding of HuMAb 5E8 to PA 63 and PA 83 using a standard ELISA assay.
  • Figure 15 is a graph showing a competitive binding assay between HuMAb 5E8 and 3 murine mAbs using PA 63 standard ELISA.
  • Figure 16 is a graph showing efficacy of HuMAbs 5E8 and 5D5 in a rabbit inhalation model.
  • Figure 17 is a graph showing efficacy of HuMAb 5E8 at lower doses in a rabbit inhalation model.
  • Figure 18 are graphs showing efficacy of HuMAb 5E8 administered post- exposure in a rabbit inhalation model.
  • A Animals treated 24 hrs and 5 days post exposure.
  • B Animals treated 48 hrs and 6 days post exposure.
  • the present mvention provides novel anti-PA antibodies and improved antibody-based therapies for treating and diagnosing anthrax (B. anthracis).
  • Methods of the invention employ isolated human monoclonal antibodies, or antigen binding portions thereof, which bind to anthrax protective antigen and inhibit the functions of anthrax protective antigen, edema factor and/or lethal factor, which methods are useful in human therapy.
  • Antibodies of the invention can be full-length (e.g., an IgGl or IgG3 antibody) or can include only an antigen-binding portion (e.g., a Fab, F(ab')2, Fv or a single chain Fv fragment).
  • the human antibodies are produced in a non-human transgenic animal, e.g., a transgenic mouse, capable of producing multiple isotypes of human monoclonal antibodies to anthrax PA (e.g., IgG, IgA and/or IgE) by undergoing V-D-J recombination and isotype switching.
  • anthrax PA e.g., IgG, IgA and/or IgE
  • particular aspects of the invention include not only antibodies, antibody fragments, and pharmaceutical compositions thereof, but also non-human transgenic animals, B-cells and hybridomas which produce monoclonal antibodies. Methods of using the antibodies of the invention to detect anthrax PA in a biological sample, are also encompassed by the invention.
  • protective antigen and PA refer to the protective antigen protein produced by the bacterium Bacillus antracis (anthrax), and include any variants, isoforms and species homologs of anthrax protective antigen, which may be naturally expressed by the bacterium or recombinantly, expressed [see Welkos et al., Gene 69: 287-300 (1988)].
  • an anti-PA antibody of the invention "neutralizes" an anthrax toxin (i.e., lethal factor or edema factor).
  • neutralizes and grammatical variations thereof, refer to an activity of an antibody of the present invention, which activity prevents entry or translocation of EF or LF into a cell susceptible to anthrax infection upon binding of the antibody to the anthrax PA.
  • binding of an antibody of the invention to anthrax PA can result in prevention of toxin translocation into a cell's cytoplasm at a number of different points during the infection process, e.g., (1) binding of anthrax PA to ATR on a cell, (2) cleavage of the PA83 to the PA63 form, (3) formation of a heptamer comprising seven PA63 units, and (4) binding of the toxin to, or otherwise associating with, the heptamer.
  • An antibody of the invention can neutralize anthrax toxin by inhibiting or blocking any one or more of the different points during the infection process through binding to anthrax PA.
  • TAA toxin neutralization assay
  • Anti-PA antibodies useful in the present invention neutralize anthrax toxin in concentrations of less than 5 ⁇ g/ml, more preferably less than 1 ⁇ g/ml, and most preferably less than 0.1 ⁇ g/ml.
  • antibodies of the invention have an ED 50 of from about 0.001 to 5 ⁇ g/ml, preferably 1 ⁇ g/ml or less, and most preferably 0.1 ⁇ g/ml or less, as measured by TNA.
  • the term "antibody” as referred to herein includes whole antibodies and any antigen binding fragment (i. e. , "antigen-binding portion") or single chain thereof.
  • An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, C HI , Cm and C H3 .
  • Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and a light chain constant region.
  • the light chain constant region is comprised of one domain, C L -
  • the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDRl, FR2, CDR2, FR3, CDR3, FR4.
  • variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • the term "antigen-binding portion" of an antibody or simply “antibody portion", as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen (e.g., B. anthracis protective antigen). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full- length antibody.
  • binding fragments which is encompassed within the term “antigen-binding portion" of an antibody, includes (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, C L and C HI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and CHI domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546), which consists of a V H domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, C L and C HI domains
  • F(ab')2 fragment a bivalent fragment comprising two Fab fragment
  • V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science
  • Such single chain antibodies are also intended to be encompassed within the term "antigen- binding portion" of an antibody.
  • antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • epitope means a protein determinant capable of specific binding to an antibody. Epitopes typically consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • bispecific molecule is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has two different binding specificities.
  • the molecule may bind to, or interact with, (a) a cell surface antigen and (b) another anti-PA antibody.
  • multispecific molecule or
  • heterospecific molecule is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has more than two different binding specificities.
  • the molecule may bind to, or interact with, (a) a cell surface antigen, (b) another anti-PA antibody, and (c) at least one other component.
  • the invention includes, but is not limited to, bispecific, trispecific, tefraspecific, and other multispecific molecules which are directed to cell surface antigens, such as B. anthracis protective antigen, and to other targets, such as another anti-PA antibody.
  • B. anthracis protective antigen such as B. anthracis protective antigen
  • targets such as another anti-PA antibody.
  • bispecific antibodies also includes diabodies.
  • Diabodies are bivalent, bispecific antibodies in which the V H and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123).
  • heteroantibodies refers to two or more antibodies, antibody binding fragments (e.g., Fab), derivatives therefrom, or antigen binding regions linked together, at least two of which have different specificities. These different specificities include a binding specificity for another anthrax antigen.
  • the term "human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germlme immunoglobulin sequences.
  • the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • human antibody is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • human monoclonal antibody refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences.
  • the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
  • recombinant human antibody is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (described further in Section I, below), (b) antibodies expressed using a recombinant expression vector transfected into a host cell, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (c) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
  • recombinant means such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (described further in Section I, below), (b) antibodies expressed using a recombinant expression vector transfected into a host cell, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (c) antibodies prepared
  • Such recombinant human antibodies have variable and constant regions derived from human ge ⁇ nline immunoglobulin sequences, hi certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V H and V regions of the recombinant antibodies are sequences that, while derived from and related to human germlme V H and V L sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • a "heterologous antibody” is defined in relation to the transgenic non-human organism producing such an antibody.
  • heterohybrid antibody refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal.
  • a heterohybrid antibody refers to an antibody having a light and heavy chains of different organismal origins.
  • an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody.
  • heterohybrid antibodies include chimeric and humanized antibodies, discussed supra.
  • an "isolated antibody,” as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that binds to protective antigen is substantially free of antibodies that bind antigens other than protective antigen).
  • An isolated antibody that binds to an epitope, isoform or variant of anthrax protective antigen may, however, have cross-reactivity to other related antigens, e.g., from other bacterial species (e.g., protective antigen species homologs).
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • a combination of "isolated" monoclonal antibodies having different specificities are combined in a well defined composition.
  • telomere binding refers to an anti-PA antibody of the present invention binding anthrax protective antigen.
  • the antibody binds with an affinity of at least about 1 x 10 7 M “1 , and binds to the protective antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the protective antigen or a closely-related antigen.
  • a non-specific antigen e.g., BSA, casein
  • the term "high affinity" for an IgG antibody refers to a binding affinity of at least about 10 7 M , preferably at least about 10 8 M _1 , more preferably at least about lO ⁇ ' ⁇ lO ⁇ M "1 , lO 1 ⁇ "1 or greater, e.g., up tol0 13 M _1 or greater.
  • high affinity binding for an IgM isotype refers to a binding affinity of at least about 1 x
  • K assoc or "K a ", as used herein, is intended to refer to the association constant of a particular antibody-antigen interaction.
  • Kdj s or "K ⁇ ”, as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction.
  • isotype refers to the antibody class (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes.
  • An isolated human antibody of the invention can be any antibody isotype, e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl,
  • isotype switching refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes.
  • nonswitched isotype refers to the isotypic class of heavy chain that is produced when no isotype switching has taken place; the CH gene encoding the nonswitched isotype is typically the first CH gene immediately downstream from the functionally rearranged VDJ gene. Isotype switching has been classified as classical or non-classical isotype switching. Classical isotype switching occurs by recombination events which involve at least one switch sequence region in the transgene.
  • Non-classical isotype switching may occur by, for example, homologous recombination between human ⁇ ⁇ and human ⁇ ⁇ ( ⁇ -associated deletion).
  • Alternative non-classical switching mechanisms such as intertransgene and/or interchromosomal recombination, among others, may occur and effectuate isotype switching.
  • switch sequence refers to those DNA sequences responsible for switch recombination.
  • a "switch donor" sequence typically a ⁇ switch region, will be 5' (i.e., upstream) of the construct region to be deleted during the switch recombination.
  • the "switch acceptor" region will be between the construct region to be deleted and the replacement constant region (e.g., ⁇ , ⁇ , etc.). As there is no specific site where recombination always occurs, the final gene sequence will typically not be predictable from the construct.
  • "glycosylation pattern” is defined as the pattern of carbohydrate units that are covalently attached to a protein, more specifically to an immunoglobulin protein.
  • a glycosylation pattern of a heterologous antibody can be characterized as being substantially similar to glycosylation patterns which occur naturally on antibodies produced by the species of the nonhuman transgenic animal, when one of ordinary skill in the art would recognize the glycosylation pattern of the heterologous antibody as being more similar to said pattern of glycosylation in the species of the nonhuman transgenic animal than to the species from which the CH genes of the transgene were derived.
  • the term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
  • the term "rearranged” as used herein refers to a configuration of a heavy chain or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete V H or V L domain, respectively.
  • a rearranged immunoglobulin gene locus can be identified by comparison to germline DNA; a rearranged locus will have at least one recombined heptamer/nonamer homology element.
  • nucleic acid molecule is intended to include DNA molecules and RNA molecules.
  • a nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • isolated nucleic acid molecule refers to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than protective antigen, which other sequences may naturally flank the nucleic acid in human genomic DNA.
  • the human anti-PA antibody or portion thereof, has the nucleotide and amino acid sequence of 5E8 (and 5E8'), 2D5, 2H4, 5D5, or heavy chain (V H ) variable regions having the nucleotide and amino acid sequences, respectively, shown in SEQ ID NOs: 1 and 2, 7 and 8, 11 and 12, and 15 and 16, or light chain (V L ) variable regions having the nucleotide and amino acid sequences, respectively, shown in SEQ ID NOs: 3 and 4, 5 and 6, 9 and 10, 13 and 14, and 17 and 18.
  • the antibodies 5E8 and 5E8' have the same heavy chain, while differing in their light chain, where 5E8 has a light chain (V L maj or ) variable region that has the nucleotide and amino acid sequences, respectively, shown in SEQ ID NOs: 3 and 4 and 5E8' has a light chain (V L m i nor ) variable region that has the nucleotide and amino acid sequences, respectively, shown in SEQ ID NOs: 5 and 6. Both of these antibodies from the 5E8 hybridoma bind to anthrax protective antigen and neutralize anthrax toxin in the TNA.
  • sequences set forth in SEQ ID NOs: 1-72 include "conservative sequence modifications", i.e., nucleotide and amino acid sequence modifications which do not significantly affect or alter the binding characteristics of the antibody encoded by the nucleotide sequence or containing the amino acid sequence.
  • conservative sequence modifications include nucleotide and amino acid substitutions, additions and deletions.
  • Modifications can be introduced into SEQ ID NOs: 1-72 by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in a human anti-PA antibody is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of an anti-PA antibody coding sequence, such as by saturation mutagenesis, and the resulting modified anti-PA antibodies can be screened for binding activity.
  • antibodies encoded by the (heavy and light chain variable region) nucleotide sequences disclosed herein and/or containing the (heavy and light chain variable region) amino acid sequences disclosed herein include substantially similar antibodies encoded by or containing similar sequences which have been conservatively modified.
  • substantially similar antibodies can be generated based on the partial (i.e., heavy and light chain variable regions) sequences disclosed herein as SEQ ID NOs: 1-18 is provided below.
  • nucleic acids the term "substantial homology" indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide or amino acid sequences can also determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been inco ⁇ orated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been inco ⁇ orated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402.
  • nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • a nucleic acid is "isolated” or "rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel elecfrophoresis and others well known in the art. See, F.
  • nucleic acid compositions of the present invention while often in a native sequence (except for modified restriction sites and the like), from either cDNA, genomic or mixtures maybe mutated, thereof in accordance with standard techniques to provide gene sequences. For coding sequences, these mutations, may affect amino acid sequence as desired.
  • DNA sequences substantially homologous to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated (where "derived" indicates that a sequence is identical or modified from another sequence).
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.
  • operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • operably linked indicates that the sequences are capable of effecting switch recombination.
  • vector as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • vector refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • recombinant expression vectors Such vectors are referred to herein as "recombinant expression vectors” (or simply, “expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retro viruses, adeno viruses and adeno-associated viruses), which serve equivalent functions.
  • the term "recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced.
  • host cell As used herein.
  • Recombinant host cells include, for example, CHO cells and lyrnphocytic cells.
  • the monoclonal antibodies (MAbs) of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology e.g. , the standard somatic cell hybridization technique of Kohler and Milstein (1975) Nature 256: 495. Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed e.g., viral or oncogenic transformation of B lymphocytes.
  • the preferred animal system for preparing hybridomas is the murine system. Hybridoma production in the mouse is a very well-established procedure, immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art.
  • Fusion partners e.g., murine myeloma cells
  • human monoclonal antibodies directed against protective antigen can be generated using transgenic mice carrying parts of the human immune system rather than the mouse system.
  • transgenic mice referred to herein as "HuMAb” mice, contain a human immunoglobulin gene miniloci that encodes unrearranged human heavy ( ⁇ and ⁇ ) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous ⁇ and K chain loci (Lonberg, et al. (1994) Nature 368(6474): 856-859).
  • mice exhibit reduced expression of mouse IgM or K, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgG ⁇ monoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113 :49- 101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N. Y. Acad. Sci 764:536-546).
  • the preparation of HuMAb mice is described in detail Section JJ below and in Taylor, L.
  • HuMAb Immunizations To generate fully human monoclonal antibodies to protective antigen, HuMAb mice can be immunized with a purified or enriched preparation of anthrax PA83 or PA63 or both and/or cells expressing protective antigen, as described by Lonberg, N. et al. (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature
  • the mice will be 6-16 weeks of age upon the first infusion.
  • a purified preparation (5-20 ⁇ g) of protective antigen (kindly supplied by Stephen Little, U.S. Army Medical Research Institute of Infectious Disease) can be used to immunize the HuMAb mice intraperitoneally.
  • Mice can also be immunized with transfected cells expressing protective antigen to promote immune responses. Cumulative experience with various antigens has shown that the HuMAb transgenic mice respond best when initially immunized intraperitoneally (JP) with antigen in complete Freund's adjuvant, followed by every other week i.p.
  • JP immunized intraperitoneally
  • mice can be immunized for each antigen. For example, a total of twelve HuMAb mice of the HC07 and HC012 strains can be immunized.
  • mice splenocytes can be isolated and fused with PEG to a mouse myeloma cell line based upon standard protocols.
  • the resulting hybridomas are then screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice are fused to one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG.
  • Cells are plated at approximately 2 x 10 5 in flat bottom microtiter plate, followed by a two week incubation in selective medium containing 20% fetal Clone Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM L- glutamine, 1 mM sodium pyruvate, 5mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and IX HAT (Sigma; the HAT is added 24 hours after the fusion). After two weeks, cells are cultured in medium in which the HAT is replaced with HT.
  • selective medium containing 20% fetal Clone Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM L- glutamine, 1 mM sodium pyruvate, 5mM HEPES, 0.055
  • Human antibodies of the invention can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (e.g., Morrison, S. (1985) Science 229:1202).
  • DNAs encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification, site directed mutagenesis) and can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences.
  • the term "operatively linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector.
  • the antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).
  • the light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the V H segment is operatively linked to the C H segment(s) within the vector and the V segment is operatively linked to the C L segment within the vector.
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell.
  • the antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
  • the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell.
  • the term "regulatory sequence” is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes.
  • regulatory sequences are described, for example, in Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenoviras major late promoter (AdMLP)) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus
  • nonviral regulatory sequences may be used, such as the ubiquitin promoter or ⁇ -globin promoter.
  • the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al).
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin or mefhotrexate, on a host cell into which the vector has been introduced.
  • Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with mefhotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • neo gene for G418 selection.
  • the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques.
  • the various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE- dextran transfection and the like.
  • Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216- 4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sha ⁇ (1982) Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells and SP2 cells.
  • Chinese Hamster Ovary CHO cells
  • dhfr- CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216- 4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sha ⁇ (1982) Mol. Biol. 159:601-621
  • another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841.
  • the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.
  • Antibodies can be recovered from the culture medium using standard protein purification methods.
  • Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V(D) J joining during B cell maturation. Germline gene sequences will also differ from the sequences of a high affinity secondary repertoire antibody at individual evenly across the variable region. For example, somatic mutations are relatively infrequent in the amino- terminal portion of framework region.
  • somatic mutations are relatively infrequent in the amino terminal portion of framework region 1 and in the carboxy- terminal portion of framework region 4. Furthermore, many somatic mutations do not significantly alter the binding properties of the antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody in order to recreate an intact recombinant antibody having binding properties similar to those of the original antibody (see PCT/US99/05535 filed on March 12, 1999, which is herein inco ⁇ orated by referenced for all purposes). Partial heavy and light chain sequence spanning the CDR regions is typically sufficient for this pu ⁇ ose. The partial sequence is used to determine which germline variable and joining gene segments contributed to the recombined antibody variable genes. The germline sequence is then used to fill in missing portions of the variable regions.
  • Heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. For this reason, it is necessary to use the corresponding germline leader sequence for expression constructs.
  • cloned cDNA sequences cab be combined with synthetic oligonucleotides by ligation or PCR amplification.
  • the entire variable region can be synthesized as a set of short, overlapping, oligonucleotides and combined by PCR amplification to create an entirely synthetic variable region clone. This process has certain advantages such as elimination or inclusion or particular restriction sites, or optimization of particular codons.
  • the nucleotide sequences of heavy and light chain transcripts from a hybridomas are used to design an overlapping set of synthetic oligonucleotides to create synthetic V sequences with identical amino acid coding capacities as the natural sequences.
  • the synthetic heavy and kappa chain sequences can differ from the natural sequences in three ways: strings of repeated nucleotide bases are interrupted to facilitate oligonucleotide synthesis and PCR amplification; optimal translation initiation sites are inco ⁇ orated according to Kozak's rules (Kozak, 1991, J. Biol. Chem. 266:19867- 19870); and, HindUI sites are engineered upstream of the translation initiation sites.
  • the optimized coding, and corresponding non-coding, strand sequences are broken down into 30 - 50 nucleotide approximately the midpoint of the corresponding non-coding oligonucleotide.
  • the oligonucleotides can be assemble into overlapping double stranded sets that span segments of 150 - 400 nucleotides. The pools are then used as templates to produce PCR amplification products of 150 -
  • variable region oligonucleotide set will be broken down into two pools which are separately amplified to generate two overlapping PCV products. These overlapping products are then combined by PCT amplification to form the complete variable region. It may also be desirable to include an overlapping fragment of the heavy or light chain constant region (including the Bbsl site of the kappa light chain, or the Agel site if the gamma heavy chain) in the PCR amplification to generate fragments that can easily be cloned into the expression vector constructs.
  • the heavy or light chain constant region including the Bbsl site of the kappa light chain, or the Agel site if the gamma heavy chain
  • the reconstructed heavy and light chain variable regions are then combined with cloned promoter, translation initiation, constant region, 3' untranslated, polyadenylation, and transcription termination, sequences to form expression vector constructs.
  • the heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected, or separately transfected into host cells which are then fused to form a host cell expressing both chains. Plasmids for use in construction of expression vectors for human IgG ⁇ are described below. The plasmids were constructed so that PCR amplified V heavy and V kappa light chain cDNA sequences could be used to reconstruct complete heavy and light chain minigenes.
  • plasmids can be used to express completely human, or chimeric IgGiK or IgG 4 ⁇ antibodies. Similar plasmids can be constructed for expression of other heavy chain isotypes, or for expression of antibodies comprismg lambda light chains.
  • a human anti-PA antibody of the invention e.g., 5E8, 2D5, 2H4, or 5D5
  • 5E8, 2D5, 2H4, or 5D5 are used to create structurally related human anti-PA antibodies that retain at least one functional property of the antibodies of the invention, such as binding to protective antigen.
  • one or more CDRs of 5E8, 2D5, 2H4, or 5D5 can be combined recombinantly with known human framework regions and CDRs to create additional, recombinantly-engineered, human anti-PA antibodies of the invention.
  • the invention provides a method for preparing an anti-PA antibody comprising: preparing an antibody comprising (1) human heavy chain framework regions and human heavy chain CDRs, wherein at least one of the human heavy chain CDRs comprises an amino acid sequence selected from the amino acid sequences of CDRs shown in Figures 1, 4, 6 or 8 (SEQ ID NOs: 20, 22, 24, 38, 40, 42, 50, 52, 54, 62, 64, and 66); and (2) human light chain framework regions and human light chain CDRs, wherein at least one of the light chain CDRs comprises an amino acid sequence selected from the amino acid sequences of CDRs shown in Figures 2, 3, 5, 7, or 9 (SEQ ID NOs: 26, 28, 30, 32, 34, 36, 44, 46, 48, 56, 58, 60, 68, 70, and 72); wherein the antibody retains the ability to bind to protective antigen.
  • the ability of the antibody to bind protective antigen can be determined using standard binding assays, such as those set forth in the Examples (e.g., an antibody binding affinity modifier
  • the recombinant antibodies of the invention prepared, as set forth above preferably comprise the heavy and light chain CDR3s of 5E8, 2D5, 2H4, or 5D5.
  • the antibodies further can comprise the CDR2s of 5E8, 2D5, 2H4 or 5D5.
  • the antibodies further can comprise the CDRls of 5E8, 2D5, 2H4 or 5D5.
  • An antibody of the invention can further comprise any combination of the CDRs. Accordingly, in another embodiment, the invention further provides anti-
  • PA antibodies comprising: (1) human heavy chain framework regions, a human heavy chain CDRl region, a human heavy chain CDR2 region, and a human heavy chain CDR3 region, wherein the human heavy chain CDR3 region is the heavy chain CDR3 of 5E8, 2D5, 2H4 or 5D5 as shown in Figures 1, 4, 6 or 8 (SEQ ID NOs: 20, 38, 50, and 62); and (2) human light chain framework regions, a human light chain CDRl region, a human light chain CDR2 region, and a human light chain CDR3 region, wherein the human light chain CDR3 region is the light chain CDR3 of 5E8, 2D5, 2H4 or 5D5 as shown in Figures 2, 3, 5, 7 or 9 (SEQ ID NO: 26, 32, 44, 56, and 68), wherein the antibody binds protective antigen.
  • the antibody may further comprise the heavy chain CDR2 and/or the light chain CDR2 of 5E8, 2D5, 2H4 or 5D5.
  • the antibody may further comprise the heavy chain CDRl and/or the light chain CDRl of 5E8, 2D5, 2H4 or 5D5.
  • the CDRl, CDR2, and/or CDR3 regions of an antibody of the invention, described above, can comprise the exact amino acid sequence(s) as those of 5E8, 2D5, 2H4 or 5D5 disclosed herein.
  • the antibodies of the invention can contain of one or more CDRs that are, for example, 95%, 98% or 99.5% identical to one or more CDRs of 5E8, 2D5, 2H4 or 5D5.
  • samples containing the antibody e.g., sera from immunized mice
  • samples containing the antibody can be tested, e.g., by ELISA.
  • ELISA e.g., by ELISA.
  • microtiter plates are coated with purified protective antigen at 20 ⁇ g/ml in PBS, and then blocked with 5% bovine serum albumin in PBS. Dilutions of plasma containing the anti-PA antibody are added to each well and incubated for 1-2 hours at 37°C.
  • mice which develop the highest titers will be used for fusions.
  • An ELISA assay as described above can also be used to screen for hybridomas that show positive reactivity with PA immunogen. Hybridomas that bind with high avidity to protective antigen will be subcloned and further characterized.
  • Clones from each hybridoma which retain the reactivity of the parent cells (by ELISA), can be chosen for making a 5-10 vial cell bank stored at -140 °C, and for antibody purification.
  • selected hybridomas can be grown in two-liter spinner-flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated before affinity chromatography with protein A-sepharose (Pharmacia, Piscataway, NJ). Eluted IgG can be checked by gel elecfrophoresis and high performance liquid chromatography to ensure purity.
  • the buffer solution can be exchanged into PBS, and the concentration can be determined by OD 280 using 1.43 extinction coefficient.
  • the monoclonal antibodies can be aliquoted and stored at -80 °C. To determine if the selected human anti-PA monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using protective antigen coated-ELISA plates as described above. Biotinylated MAb binding can be detected with a strep-avidin-alkaline phosphatase probe. To determine the isotype of purified antibodies, isotype ELISAs can be performed.
  • wells of microtiter plates can be coated with 10 ⁇ g/ml of anti- human Ig overnight at 4°C. After blocking with 5% BSA, the plates are reacted with 10 ⁇ g/ml of monoclonal antibodies or purified isotype controls, at ambient temperature for two hours. The wells can then be reacted with either human IgGl or human IgM-specific alkaline phosphatase-conjugated probes. Plates are developed and analyzed as described above. Anti-PA human IgGs can be further tested for reactivity with protective antigen by Western blotting. For example, PA can be subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel elecfrophoresis.
  • SDS sodium dodecyl sulfate
  • the separated antigens will be transferred to nitrocellulose membranes, blocked with 20% mouse serum, and probed with the monoclonal antibodies to be tested.
  • Human IgG binding can be detected using anti-human IgG alkaline phosphatase and developed with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).
  • the invention provides transgenic and franschromosomal nonhuman animals, such as transgenic or transchromosomal mice, which are capable of expressing human monoclonal antibodies that specifically bind to protective antigen.
  • the invention provides a transgenic or transchromosomal mouse having a genome comprising a human heavy chain transgene, such that the mouse produces human anti-PA antibodies when immunized with anthrax protective antigen and/or cells expressing protective antigen.
  • the human heavy chain transgene can be integrated into the chromosomal DNA of the mouse, as is the case for transgenic, e.g., HuMAb Mouse ® , as described in detail and exemplified herein.
  • the human heavy chain transgene can be maintained exfrachromosomally, as is the case for transchromosomal (e.g., KM-Mouse ® ) mice as described in WO 02/43478.
  • Such transgenic and transchromosomal animals are capable of producing multiple isotypes of human monoclonal antibodies to protective antigen (e.g., IgG, IgA and/or IgE) by undergoing V-D-J recombination and isotype switching.
  • Isotype switching may occur by, e.g., classical or non-classical isotype switching.
  • the design of a transgenic or transchromosomal nonhuman animal that responds to foreign antigen stimulation with a heterologous antibody repertoire requires that the heterologous immunoglobulin transgenes contained within the transgenic animal function correctly throughout the pathway of B-cell development. This includes, for example, isotype switching of the heterologous heavy chain transgene.
  • transgenes are constructed so as to produce isotype switching and one or more of the following of antibodies: (1) high level and cell-type specific expression, (2) functional gene rearrangement, (3) activation of and response to allelic exclusion, (4) expression of a sufficient primary repertoire, (5) signal transduction, (6) somatic hypermutation, and (7) domination of the transgene antibody locus during the immune response. Not all of the foregoing criteria need be met. For example, in those embodiments wherein the endogenous immunoglobulin loci of the transgenic animal are functionally disrupted, the transgene need not activate allelic exclusion.
  • the transgene comprises a functionally rearranged heavy and/or light chain immunoglobulin gene
  • the second criteria of functional gene rearrangement is unnecessary, at least for that transgene which is already rearranged.
  • the transgenic or transchromosomal nonhuman animals which can be used to generate human monoclonal antibodies of the invention contain rearranged, unrearranged or a combination of rearranged and unrearranged heterologous immunoglobulin heavy and light chain transgenes in the germline of the transgenic animal.
  • Each of the heavy chain transgenes comprises at least one C H gene.
  • the heavy chain transgene may contain functional isotype switch sequences, which are capable of supporting isotype switching of a heterologous transgene encoding multiple C H genes in the B-cells of the transgenic animal.
  • switch sequences may be those which occur naturally in the germline immunoglobulin locus from the species that serves as the source of the transgene CH genes, or such switch sequences maybe derived from those which occur in the species that is to receive the transgene construct (the transgenic animal).
  • a human transgene construct that is used to produce a transgenic mouse may produce a higher frequency of isotype switching events if it inco ⁇ orates switch sequences similar to those that occur naturally in the mouse heavy chain locus, as presumably the mouse switch sequences are optimized to function with the mouse switch recombinase enzyme system, whereas the human switch sequences are not.
  • Switch sequences may be isolated and cloned by conventional cloning methods, or may be synthesized de novo from overlapping synthetic oligonucleotides designed on the basis of published sequence information relating to immunoglobulin switch region sequences (Mills et al, Nucl. Acids Res. 15:7305-7316 (1991); Sideras et al.
  • the transgenes used to generate the transgenic nonhuman animals used to produce the human monoclonal antibodies of the invention include a heavy chain transgene comprising DNA encoding at least one variable gene segment, one diversity gene segment, one joining gene segment and at least one constant region gene segment.
  • the immunoglobulin light chain transgene comprises DNA encoding at least one variable gene segment, one joining gene segment and at least one constant region gene segment.
  • the gene segments encoding the light and heavy chain gene segments are heterologous to the transgenic animal in that they are derived from, or correspond to, DNA encoding immunoglobulin heavy and light chain gene segments from a species not consisting of the transgenic nonhuman animal.
  • the transgene is constructed such that the individual gene segments are unrearranged, i.e., not rearranged so as to encode a functional immunoglobulin light or heavy chain.
  • Such unrearranged transgenes support recombination of the V, D, and J gene segments (functional rearrangement) and preferably support inco ⁇ oration of all or a portion of a D region gene segment in the resultant rearranged immunoglobulin heavy chain within the transgenic animal when exposed to Protective antigen.
  • the transgenes comprise an unrearranged
  • transgenes typically comprise a substantial portion of the C, D, and J segments as well as a subset of the V gene segments.
  • the various regulatory sequences e.g., promoters, enhancers, class switch regions, splice- donor and splice-acceptor sequences for RNA processing, recombination signals and the like, comprise corresponding sequences derived from the heterologous DNA.
  • Such regulatory sequences may be inco ⁇ orated into the transgene from the same or a related species of the nonhuman animal used in the invention.
  • human immunoglobulin gene segments may be combined in a transgene with a rodent immunoglobulin enhancer sequence for use in a transgenic mouse.
  • synthetic regulatory sequences may be inco ⁇ orated into the transgene, wherein such synthetic regulatory sequences are not homologous to a functional DNA sequence that is known to occur naturally in the genomes of mammals.
  • Synthetic regulatory sequences are designed according to consensus rules, such as, for example, those specifying the permissible sequences of a splice-acceptor site or a promoter/enhancer motif.
  • a minilocus comprises a portion of the genomic immunoglobulin locus having at least one internal (i.e., not at a terminus of the portion) deletion of a non-essential DNA portion (e.g., intervening sequence; intron or portion thereof) as compared to the naturally-occurring germline Ig locus.
  • the transgenic or transchromosomal animal used to generate human antibodies to protective antigen contains at least one, typically 2-10, and sometimes 25-50 or more copies of the transgene described in Example 12 of WO 98/24884 (e.g., pHCl or pHC2) bred with an animal containing a single copy of a light chain transgene described in Examples 5, 6, 8, or 14 of WO 98/24884, and the offspring bred with the J H deleted animal described in Example 10 of WO 98/24884. Animals are bred to homozygosity for each of these three traits.
  • Such animals have the following genotype: a single copy (per haploid set of chromosomes) of a human heavy chain unrearranged mini-locus (described in Example 12 of WO 98/24884), a single copy (per haploid set of chromosomes) of a rearranged human K light chain construct (described in Example 14 of WO 98/24884), and a deletion at each endogenous mouse heavy chain locus that removes all of the functional J H segments (described in Example 10 of WO 98/24884).
  • Such animals are bred with mice that are homozygous for the deletion of the J H segments (Example 10 of WO
  • the B cells will be monospecific with regards to the human or mouse light chains because expression of the single copy of the rearranged human K light chain gene will allelically and isotypically exclude the rearrangement of the endogenous mouse K and lambda chain genes in a significant fraction of B-cells.
  • Preferred transgenic and transchromosomal nonhuman animals e.g., mice, will exhibit immunoglobulin production with a significant repertoire, ideally substantially similar to that of a native mouse.
  • the total immunoglobulin levels will range from about 0.1 to 10 mg/ml of serum, preferably 0.5 to 5 mg/ml, ideally at least about 1.0 mg/ml.
  • the adult mouse ratio of serum IgG to IgM is preferably about 10:1.
  • the IgG to IgM ratio will be much lower in the immature mouse, hi general, greater than about 10%, preferably 40 to 80% of the spleen and lymph node B cells express exclusively human IgG protein.
  • the repertoire will ideally approximate that shown in a native mouse, usually at least about 10% as high, preferably 25 to 50% or more.
  • immimoglobulins ideally IgG
  • 10 4 to 10 6 or more will be produced, depending primarily on the number of different V, J and D regions introduced into the mouse genome.
  • immunoglobulins will typically recognize about one-half or more of highly antigenic proteins, e.g., staphylococcus protein A. Typically, the immunoglobulins will exhibit an affinity (K D ) for preselected antigens of below 10 "7 M, such as of below 10 "8 M, 10 "9 M or 10 "10 M, or even lower. In some embodiments, it may be preferable to generate nonhuman animals with predetermined repertoires to limit the selection of V genes represented in the antibody response to a predetermined antigen type.
  • a heavy chain transgene having a predetermined repertoire may comprise, for example, human VH genes which are preferentially used in antibody responses to the predetermined antigen type in humans.
  • VH genes may be excluded from a defined repertoire for various reasons (e.g., have a low likelihood of encoding high affinity V regions for the predetermined antigen; have a low propensity to undergo somatic mutation and affinity sha ⁇ ening; or are immunogenic to certain humans).
  • a transgene containing various heavy or light chain gene segments such gene segments may be readily identified, e.g., by hybridization or DNA sequencing, as being from a species of organism other than the transgenic animal.
  • Transgenic and transchromosomal nonhuman animals e.g., mice, as described above can be immunized with, for example, a purified or recombinant preparation of Protective antigen and/or cells expressing protective antigen.
  • the transgenic animals can be immunized with DNA encoding human protective antigen.
  • the animals will then produce B cells which undergo class- switching via intratransgene switch recombination (cis-switching) and express immunoglobulins reactive with protective antigen.
  • the immunoglobulins can be human antibodies (also referred to as "human sequence antibodies”), wherein the heavy and light chain polypeptides are encoded by human transgene sequences, which may include sequences derived by somatic mutation and V region recombinatorial joints, as well as germline-encoded sequences; these human antibodies can be referred to as being substantially identical to a polypeptide sequence encoded by a human V L or V H gene segment and a human J L or D H and J H segment, even though other non-germline sequences may be present as a result of somatic mutation and differential V-J and V-D-J recombination joints.
  • variable regions of each antibody chain are typically at least 80 percent encoded by human germline V, J, and, in the case of heavy chains, D, gene segments; frequently at least 85 percent of the variable regions are encoded by human germline sequences present on the transgene; often 90 or 95 percent or more of the variable region sequences are encoded by human germline sequences present on the fransgene.
  • non-germline sequences are introduced by somatic mutation and VJ and VDJ joining, the human sequence antibodies will frequently have some variable region sequences (and less frequently constant region sequences) which are not encoded by human V, D, or J gene segments as found in the human transgene(s) in the germline of the mice.
  • non-germline sequences will cluster in or near CDRs, or in regions where somatic mutations are known to cluster.
  • Human antibodies which bind to the predetermined antigen can result from isotype switching, such that human antibodies comprising a human sequence ⁇ chain (such as ⁇ l, ⁇ 2a, ⁇ 2B, or ⁇ 3) and a human sequence light chain (such as kappa) are produced.
  • Such isotype-switched human antibodies often contain one or more somatic mutation(s), typically in the variable region and often in or within about 10 residues of a CDR) as a result of affinity maturation and selection of B cells by antigen, particularly subsequent to secondary (or subsequent) antigen challenge.
  • high affinity human antibodies may have binding affinities (K D ) of below 10 "7 M, such as of below 10 "8 M, 10 "9 M or 10 "10 M, or even lower.
  • Another aspect of the invention includes B cells derived from transgenic or transchromosomal nonhuman animals as described herein. The B cells can be used to generate hybridomas expressing human monoclonal antibodies which bind with high affinity (e.g., lower than 10 "7 M) to human protective antigen.
  • the invention provides a hybridoma which produces a human antibody having an affinity (K D ) of below 10 "7 M, such as of below 10 "8 M, 10 "9 M or 10 "10 M, or even lower when determined by surface plasmon resonance (SPR) technology in a
  • the antibody comprises: a human sequence light chain composed of (1) a light chain variable region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human V L gene segment and a human J L segment, and (2) a light chain constant region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human C L gene segment; and a human sequence heavy chain composed of a (1) a heavy chain variable region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human V H gene segment, optionally a D region, and a human JH segment, and (2) a constant region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human C H gene segment.
  • V region transgene is a yeast artificial chromosome comprising a portion of a human V H or V L (V K ) gene segment array, as may naturally occur in a human genome or as may be spliced together separately by recombinant methods, which may include out-of-order or omitted V gene segments.
  • V gene segments are contained on the YAC.
  • the V repertoire expansion method wherein the animal expresses an immunoglobulin chain comprising a variable region sequence encoded by a V region gene segment present on the V region transgene and a C region encoded on the human Ig transgene.
  • V repertoire expansion method transgenic animals having at least 5 distinct V genes can be generated; as can animals containing at least about 24 V genes or more.
  • Some V gene segments may be non-functional (e.g., pseudogenes and the like); these segments may be retained or may be selectively deleted by recombinant methods available to the skilled artisan, if desired.
  • the trait can be propagated and bred into other genetic backgrounds, including backgrounds where the functional YAC having an expanded V segment repertoire is bred into a nonhuman animal germline having a different human Ig transgene.
  • Multiple functional YACs having an expanded V segment repertoire may be bred into a germline to work with a human Ig transgene (or multiple human Ig transgenes).
  • YAC transgenes when integrated into the genome may substantially lack yeast sequences, such as sequences required for autonomous replication in yeast; such sequences may optionally be removed by genetic engineering (e.g., restriction digestion and pulsed-field gel elecfrophoresis or other suitable method) after replication in yeast is no longer necessary (i.e., prior to introduction into a mouse ES cell or mouse prozygote).
  • Methods of propagating the trait of human sequence immunoglobulin expression include breeding a transgenic animal having the human Ig transgene(s), and optionally also having a functional YAC having an expanded V segment repertoire. Both V H and V L gene segments may be present on the YAC.
  • the transgenic animal may be bred into any background desired by the practitioner, including backgrounds harboring other human transgenes, including human Ig transgenes and/or transgenes encoding other human lymphocyte proteins.
  • the invention also provides a high affinity human sequence immunoglobulin produced by a transgenic mouse having an expanded V region repertoire YAC transgene.
  • Transgenic animals containing a rearranged heavy and rearranged light immunoglobulin transgene are of these categories of transgenic animal, the order of preference is as follows LI > I > HI > IV where the endogenous light chain genes (or at least the K gene) have been knocked out by homologous recombination (or other method) and I > II > HI >IV where the endogenous light chain genes have not been knocked out and must be dominated by allelic exclusion.
  • human monoclonal antibodies to protective antigen, or antigen-binding portions thereof can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., an Fab' fragment) to generate a bispecific or multispecific molecule which binds to multiple binding sites or target epitopes.
  • another functional molecule e.g., another peptide or protein (e.g., an Fab' fragment) to generate a bispecific or multispecific molecule which binds to multiple binding sites or target epitopes.
  • an antibody or antigen-binding portion of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic.
  • the present invention includes bispecific and multispecific molecules comprising at least one first binding specificity for protective antigen and a second binding specificity for a second target epitope.
  • the bispecific and multispecific molecules of the invention comprise as a binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., an Fab, Fab', F(ab')2, Fv, or a single chain Fv.
  • the antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in Ladner et al. U.S. Patent No. 4,946,778, issued August 7, 1990, the contents of which is expressly inco ⁇ orated by reference.
  • human monoclonal antibodies are preferred, other antibodies which can be employed in the bispecific or multispecific molecules of the invention are murine, chimeric and humanized monoclonal antibodies.
  • Chimeric mouse-human monoclonal antibodies i.e., chimeric antibodies
  • a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted,
  • restriction enzymes for example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted,
  • the chimeric antibody can be further humanized by replacing sequences of the Fv variable region which are not directly involved in antigen binding with equivalent sequences from human Fv variable regions.
  • General reviews of humanized chimeric antibodies are provided by Morrison, S. L., 1985, Science 229: 1202-1207 and by Oi et al, 1986, BioTechniques 4:214. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from 7E3, an anti-GPH ⁇ HIa antibody producing hybridoma.
  • Suitable humanized antibodies can alternatively be produced by CDR substitution U.S. Patent 5,225,539; Jones et al. 1986 Nature 321 :552- 525; Verhoeyan et al. 1988 Science 239:1534; and Beidler et al. 1988 J. Immunol. 141:4053-4060. All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to the Fc receptor.
  • An antibody can be humanized by any method, which is capable of replacing at least a portion of a CDR of a human antibody with a CDR derived from a non-human antibody.
  • Winter describes a method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed March 26, 1987), the content of which is expressly inco ⁇ orated by reference.
  • the human CDRs may be replaced with non-human CDRs using oligonucleotide site- directed mutagenesis as described in International Application WO 94/10332 entitled, Humanized Antibodies to Fc Receptors for Immunoglobulin G on Human Mononuclear Phagocytes.
  • chimeric and humanized antibodies in which specific amino acids have been substituted, deleted or added have amino acid substitutions in the framework region, such as to improve binding to the antigen.
  • amino acids located in the human framework region can be replaced with the amino acids located at the corresponding positions in the mouse antibody. Such substitutions are known to improve binding of humanized antibodies to the antigen in some instances.
  • Antibodies in which amino acids have been added, deleted, or substituted are referred to herein as modified antibodies or altered antibodies.
  • modified antibody is also intended to include antibodies, such as monoclonal antibodies, chimeric antibodies, and humanized antibodies which have been modified by, e.g., deleting, adding, or substituting portions of the antibody.
  • an antibody can be modified by deleting the constant region and replacing it with a constant region meant to increase half-life, e.g., serum half-life, stability or affinity of the antibody. Any modification is within the scope of the invention so long as the bispecific and multispecific molecule has at least one antigen binding region specific for an Fc ⁇ R and triggers at least one effector function.
  • Bispecific and multispecific molecules of the present invention can be made using chemical teclmiques (see e.g., D. M. Kranz et al. (1981) Proc.
  • bispecific and multispecific molecules of the present mvention can be prepared by conjugating the constituent binding specificities, e.g., the anti-FcR and anti-PA binding specificities, using methods known in the art and described in the examples provided herein. For example, each binding specificity of the bispecific and multispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation.
  • cross-linking agents examples include protein A, carbodiimide, N-succinimidyl-S-acetyl- thioacetate (SAT A), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o- phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate (sulfo- SMCC) (see e.g., Ka ⁇ ovsky et al. (1984) J. Exp. Med.
  • Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, EL).
  • the binding specificities are antibodies (e.g., two humanized antibodies), they can be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains.
  • the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.
  • both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific and multispecific molecule is a MAb x MAb, MAb x Fab, Fab x F(ab')2 or ligand x Fab fusion protein.
  • a bispecific and multispecific molecule of the invention e.g., a bispecific molecule can be a single chain molecule, such as a single chain bispecific antibody, a single chain bispecific molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprismg two binding determinants.
  • Bispecific and multispecific molecules can also be single chain molecules or may comprise at least two single chain molecules. Methods for preparing bi- and multspecific molecules are described for example in U.S. Patent Number 5,260,203; U.S. Patent Number 5,455,030; U.S. Patent Number 4,881,175; U.S. Patent Number 5,132,405; U.S. Patent Number 5,091,513; U.S.
  • a labeled reagent e.g., an antibody
  • the FcR-antibody complexes can be detected using e.g., an enzyme-linked antibody or antibody fragment which recognizes and specifically binds to the antibody-FcR complexes.
  • the complexes can be detected using any of a variety of other immunoassays.
  • the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is inco ⁇ orated by reference herein).
  • the radioactive isotope can be detected by such means as the use of a ⁇ counter or a scintillation counter or by autoradiography.
  • compositions in another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, containing one or a combination of human anti-PA monoclonal antibodies, or antigen-binding portion(s) thereof, formulated together with a pharmaceutically acceptable carrier.
  • the compositions include a combination of multiple (e.g., two or more) isolated human anti-PA antibodies or antigen-binding portions thereof of the invention.
  • each of the antibodies or antigen-binding portions thereof of the composition binds to a distinct, pre-selected epitope of protective antigen.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the active compound i.e., antibody, bispecific and multispecific molecule, maybe coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
  • a “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like
  • nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • a composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
  • the active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • To administer a compound of the invention by certain routes of administration it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
  • the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al (1984) J Neuroimmunol. 7:27).
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be inco ⁇ orated into the compositions.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, hi many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by including in the composition an agent that delays abso ⁇ tion, for example, monostearate salts and gelatin.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfilfration.
  • dispersions are prepared by inco ⁇ orating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above, hi the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze- drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response).
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydrox toluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydrox toluene (BHT), le
  • formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect.
  • compositions of this invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration of compositions of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged abso ⁇ tion of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay abso ⁇ tion such as aluminum monostearate and gelatin.
  • the compounds of the present invention When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.01 to 99.5% (more preferably, 0.1 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier. Regardless of the route of administration selected, the compounds of the present mvention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • a suitable daily dose of a compositions of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • Any mode of parenteral administration is suitable for use in the present invention. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the site of the target.
  • the effective daily dose of a therapeutic compositions may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).
  • Therapeutic compositions can be administered with medical devices known in the art. For example, in a prefened embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556.
  • Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent
  • the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes.
  • liposomes For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331.
  • the liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade (1989) J Clin. Pharmacol. 29:685).
  • exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low et al); mannosides (Umezawa et al, (1988) Biochem.
  • the therapeutic compounds of the invention are formulated in liposomes; in a more preferred embodiment, the liposomes include a targeting moiety.
  • the therapeutic compounds in the liposomes are delivered by bolus injection to a site proximal to the desired area, e.g., the site of inflammation or infection.
  • the composition must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • a therapeutically effective amount of an antibody of the invention is an amount sufficient to prevent death in a subject exposed to anthrax.
  • the ability of an antibody of the invention to reduce signs and/or symptoms, including preventing death, associated with anthrax infection can be evaluated in an animal model system predictive of efficacy of the antibody in treating human anthrax infection.
  • Examples 6-8, infra which provide a rabbit studies where animals were infected with anthrax and then treated with an antibody of the invention.
  • a therapeutically effective amount of an antibody of the invention can be evaluated by examining the antibody's ability to neutralize an anthrax toxin in vitro in a toxin neutralization assay, which is well known in the art and described supra.
  • One of ordinary skill in the art would be able to determine such therapeutically effective amounts based on factors such as the subject's size, the severity of the signs and/or subject's symptoms, and the particular composition or route of administration selected.
  • compositions containing an antibody of the invention for therapeutic use must be sterile and fluid to the extent that the composition is deliverable by syringe.
  • the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.
  • the injectable compositions can be brought about by including in the composition an agent which delays abso ⁇ tion, for example, aluminum monostearate or gelatin.
  • an agent which delays abso ⁇ tion for example, aluminum monostearate or gelatin.
  • the active compound when suitably protected, as described above, the compound may be orally administered, for example, with an inert diluent or an assimilable edible carrier.
  • the human antibodies, antibody compositions and methods of the present invention have numerous in vitro and in vivo diagnostic and therapeutic utilities involving the diagnosis, prevention and treatment of anthrax infection.
  • these antibodies can be administered to human subjects to treat and/or prevent anthrax infection.
  • a blood or tissue sample can be removed from a subject and contacted with an antibody of the invention under conditions which allow detection of anthrax PA in the sample in order to diagnose an anthrax infection in the subject.
  • the term "subject" is intended to mclude human and non-human animals.
  • the antibodies are used in vivo to treat, prevent or diagnose anthrax infection, hi one prophylactic use, the subject has not been exposed to anthrax, and therefore can undergo a prophylactic-preexposure treatment with a human antibody of the invention to prevent infection by anthrax.
  • a prophylactic-preexposure treatment with a human antibody of the invention to prevent infection by anthrax.
  • a subject known to have been exposed to anthrax, but who does not display signs or symptoms of disease can undergo a post-exposure prophylactic treatment to prevent the pathology associated with anthrax disease progression.
  • a therapeutic freatment the subject has been exposed to anthrax, is infected and exhibits signs and/or symptoms of the disease.
  • Antibodies of the invention can be used in both of the prophylactic settings and in therapeutic treatment of anthrax.
  • the human antibodies, antibody compositions and methods of the present invention can be used to treat a subject which has been (or suspected of having been) infected with B. anthracis and/or displays signs and/or symptoms of anthrax infection. Signs and/or symptoms of a subject infected with anthrax, or potentially infected, are measurable.
  • signs can include low pO 2 (oxygen in blood), elevated body temperature (measured fever), adventitious sounds on lung exam, low blood pressure (or other signs of shock), widened mediastinum on chest X ray (e.g., due to lysis of lymphnodes draining the lungs), or other signs typically known to be associated with uncontrolled pulmonary anthrax infection and toxin release; symptoms can include shortness of breath, cough, chills, feeling feverish, weakness, pain with deep breath, or other symptoms generally associated with pulmonary anthrax. Cutaneous anthrax begins as a pruritic papule or vesicle that enlarges and erodes (1-2 days) leaving a necrotic ulcer with subsequent formation of a central black eschar.
  • Gastrointestinal anthrax may result in pharyngeal lesions with sore throat, dypshagia marked neck swelling and regional lymphadenopathy, or intestinal infection characterized by fever, severe abdominal pain, massive ascites, hematemesis, and bloody diarrhea.
  • hemonhagic meningitis can result from hematogenous spread of the organism from the primary site.
  • the antibodies e.g., human monoclonal antibodies, multispecific and bispecific molecules and compositions
  • the antibodies can be used to detect levels of protective antigen in a biological sample (e.g., in blood from an anthrax- infected subject), which levels can then be linked to certain disease symptoms.
  • the antibodies (e.g., human antibodies, multispecific and bispecific molecules and compositions) of the invention can be initially tested for binding activity associated with therapeutic or diagnostic use in vitro.
  • compositions of the invention can be tested using the ELISA and flow cytometric assays described in the Examples below.
  • the antibodies (e.g., human antibodies, multispecific and bispecific molecules and compositions) of the invention have additional utility in therapy and diagnosis of anthrax.
  • the human monoclonal antibodies, the multispecific or bispecific molecules can be used to elicit in vivo or in vitro one or more of the following biological activities: (1) prevent entry or translocation of anthrax toxin into the cell; (2) prevent binding of protective antigen to ATR on cells that express ATR; (3) inhibit cleavage of PA83 to PA63; (4) prevent formation of the PA heptamer; (5) block or reduce binding of a toxin (edema factor or lethal factor) to the heptamer; (6) neutralize lethal factor or edema factor such that the toxins are unable to cause physiological damage to the cell; and/or (7) otherwise protect cells against the lethal effects of toxins.
  • a toxin edema factor or lethal factor
  • Suitable routes of administering the antibody compositions (e.g., human monoclonal antibodies, multispecific and bispecific molecules and immunoconjugates ) of the invention in vivo and in vitro are well known in the art and can be selected by those of ordinary skill.
  • the antibody compositions can be administered by injection (e.g., intravenous or subcutaneous) as described supra. Suitable dosages of the molecules used will depend on the age and weight of the subject and the concentration and/or formulation of the antibody composition.
  • treatment i.e., infection with anthrax and showing clinical signs and/or symptoms
  • prophylactic treatment e.g., prevention of clinical manifestations of anthrax such as neutropenia, clinical signs and/or clinical symptoms
  • the skilled practitioner may administer from 0.3 mg/kg to 50 mg/kg or from about 1 mg/kg to 12 mg/kg.
  • the dosage will depend on, inter alia, the health of the patient, whether infection by anthrax is present, whether signs or symptoms of anthrax disease are present, and whether administration is for prophylaxis.
  • the skilled practioner will appreciate that dosaging of an antibody of the invention can be modified depending on these factors. The following dosaging regimens should, therefore, not be construed as limiting.
  • substantially high dose can be administered, e.g., at least 25 mg/kg, or 50 mg/kg or even 100 mg/kg, in order to save the patients life.
  • a patient who is believed to be infected but exhibits no signs or symptoms can benefit from relatively lower dosage, e.g, less than 12-15 mg/kg or even 1-3 mg/kg.
  • the practioner can use intennediate dosage ranges, e.g., 12-50 mg/kg.
  • Human anti-PA antibodies of the invention can be co-administered with one or more other therapeutic or immunostimulatory agents.
  • the antibody can be administered before, after or concunently with the agent or can be co-administered with other known therapies including Anthrax vaccines, antibodies against LF, EF, PA, and B. anthracis antibiotics, e.g., ciprofloxacin, doxycycline, chloramphenicol, clindamycin, tetracycline, rifampin, and vancomycin.
  • the invention provides methods for detecting the presence of protective antigen in a sample, or measuring the amount of anthrax protective antigen, comprising contacting the sample, and a control sample, with a human monoclonal antibody of the invention, or an antigen binding portion thereof, which specifically binds to protective antigen, under conditions that allow for formation of a complex between the antibody or portion thereof and protective antigen. The formation of a complex is then detected, wherein a difference between complex formation of the sample compared to the control sample is indicative the presence of protective antigen in the sample.
  • kits comprising the antibody compositions of the mvention (e.g., human antibodies and immunoconjugates) and instructions for use.
  • the kit can further contain one or more additional diagnostic, therapeutic or immunostimulatory agents as described above.
  • the present invention is further illustrated by the following examples which should not be construed as further limiting.
  • the invention also provides methods of screening to identify new antibodies having properties that make them useful in methods of treatment.
  • Antibodies which are particularly useful in methods of treatment, in addition to having the neutralization activity as described supra, and in the Examples infra, include antibodies having one or more of the following characteristics: binds to Fc receptor for neutralization activity (e.g., as measured by use of Fc receptor blocking antibodies such as 2.4G2 or use of Fab ' or ScFv in TNA with at least 5-fold, more preferably 10-fold, reduction in activity); exhibits higher affinity for PA 63 over PA 83 (e.g., as measured by standard ELISA using 1 ⁇ g/ml antibody), does not bind PA 83 at 1 ⁇ g/ml, OD 405nrn 0.2 or less according to standard ELISA, does not block anthrax lethal factor/edema factor according to competition binding assay described in Little et al., 1996, Microbiology 142:707-715, or does not compete with an anti-PA antibody selected from murine 1G3, murine 2D5 and murine 14B7 in binding to PA 63 (e
  • Antibodies having many or all of these characteristics are desirable for in vivo use. Particularly useful antibodies do not compete with an anti-PA antibody selected from murine 14B7 in binding to PA 63 (see Little et al, 1996, Microbiology 142:707-715), i.e., antibodies which block PA binding to ATR, and are desirable for in vivo therapy. hi a particular screening method of the invention, antibodies are selected based on (1) first selecting one or more antibodies which neutralizes anthrax toxin in a toxin neutralization assay, and (2) then selecting an antibody which has an ED 50 of 0.1 ⁇ g/ml or less in a TNA.
  • an antigen-antibody binding affinity assay (e.g., ELISA) is not used prior to the TNA steps. This is in contrast with methods conventionally used in the prior art, which rely on antigen-antibody binding affinity assays as a first and primary method of screening. By including such a step many antibodies, which would otherwise demonstrate excellent toxin neutralizing activity, such as HuMAb 5E8, are excluded from further development.
  • the present inventors have discovered that binding affinity to PA is not a useful criteria, and therefore it is omitted from selection methods of the present invention. Additional, selection steps can be practiced in addition to the two steps described above to obtain antibodies having the properties described.
  • Example 1 Preparation of transgenic animals which express fully human antibodies
  • the plasmid pICEmu contains an EcoRI/XhoI fragment of the murine Ig heavy chain locus, spanning the mu gene, that was obtained from a Balb/C genomic lambda phage library (Marcu et al. Cell 22: 187, 1980). This genomic fragment was subcloned into the XhoI/EcoRI sites of the plasmid pICEMM ⁇ (Marsh et al; Gene 32, 481-485, 1984).
  • the heavy chain sequences included in pICEmu extend downstream of the EcoRI site located just 3' of the mu intronic enhancer, to the Xhol site located approximately 1 kb downstream of the last transmembrane exon of the mu gene; however, much of the mu switch repeat region has been deleted by passage in E. coli.
  • the targeting vector was constructed as follows. A 1.3 kb HindlH/Smal fragment was excised from pICEmu and subcloned into HindHI/Smal digested pBluescript (Stratagene, La JoUa, CA). This pICEmu fragment extends from the HindHI site located approximately 1 kb 5' of Cmul to the Smal site located within Cmul.
  • the resulting plasmid was digested with Smal/Spel and the approximately 4 kb Smal/Xbal fragment from pICEmu, extending from the Smal site in Cmul 3' to the Xbal site located just downstream of the last Cmu exon, was inserted.
  • the resulting plasmid, pTARl was linearized at the Smal site, and a neo expression cassette inserted.
  • This cassette consists of the neo gene under the transcriptional confrol of the mouse phosphoglycerate kinase (pgk) promoter (Xbal/Taql fragment; Adra et al.
  • the neo cassette was excised from pGEM-7 (KJ1) by EcoRI/Sall digestion, blunt ended and subcloned into the Smal site of the plasmid pTARl, in the opposite orientation of the genomic Cmu sequences.
  • the resulting plasmid was linearized with Not I, and a he ⁇ es simplex virus thymidine kinase (tk) cassette was inserted to allow for enrichment of ES clones bearing homologous recombinants, as described by Mansour et al. (1988) Nature 336: 348-352.
  • This cassette consists of the coding sequences of the tk gene bracketed by the mouse pgk promoter and polyadenylation site, as described by Tybulewicz et al. (1991) Cell 65: 1153-1163.
  • the resulting CMD targeting vector contains a total of approximately 5.3 kb of homology to the heavy chain locus and is designed to generate a mutant mu gene into which has been inserted a neo expression cassette in the unique Smal site of the first Cmu exon.
  • the targeting vector was linearized with Pvul, which cuts within plasmid sequences, prior to electroporation into ES cells.
  • AB-1 ES cells (McMahon, A. P. and Bradley, A., (1990) Cell 62: 1073- 1085) were grown on mitotically inactive SNL76/7 cell feeder layers (ibid.) essentially as described (Robertson, E. J. (1987) in Teratocarcinomas and Embryonic Stem Cells: a Practical Approach (E. J. Robertson, ed.) Oxford: IRL Press, p. 71-112).
  • the linearized CMD targeting vector was electroporated into AB-1 cells by the methods described Hasty et al. (Hasty, P. R. et al. (1991) Nature 350: 243-246).
  • Electroporated cells were plated into 100 mm dishes at a density of 1-2 x 10 ⁇ cells/dish. After 24 hours, G418 (200 micrograms/ml of active component) and FIAU (5 x 10 "7 M) were added to the medium, and drug-resistant clones were allowed to develop over 8-9 days. Clones were picked, trypsinized, divided into two portions, and further expanded. Half of the cells derived from each clone were then frozen and the other half analyzed for homologous recombination between vector and target sequences. DNA analysis was carried out by Southern blot hybridization. DNA was isolated from the clones as described Laird et al. (Laird, P. W. et al, (1991) Nucleic
  • Isolated genomic DNA was digested with Spel and probed with a 915 bp Sad fragment, probe A (see Figure 1), which hybridizes to a sequence between the mu intronic enhancer and the mu switch region.
  • Probe A detects a 9.9 kb Spel fragment from the wild type locus, and a diagnostic 7.6 kb band from a mu locus which has homologously recombined with the CMD targeting vector (the neo expression cassette contains a Spel site).
  • 3 displayed the 7.6 kb Spel band indicative of homologous recombination at the mu locus.
  • mice bearing the mutated mu gene The three targeted ES clones, designated number 264, 272, and 408, were thawed and injected into C57BL/6J blastocysts as described by Bradley (Bradley, A. (1987) in Teratocarcinomas and Embryonic Stem Cells: a Practical Approach. (E. J. Robertson, ed.) Oxford: IRL Press, p. 113-151). Injected blastocysts were transferced into the uteri of pseudopregnant females to generate chimeric mice representing a mixture of cells derived from the input ES cells and the host blastocyst.
  • ES cell contribution to the chimera can be visually estimated by the amount of agouti coat coloration, derived from the ES cell line, on the black C57BL/6J background.
  • Clones 272 and 408 produced only low percentage chimeras (i.e. low percentage of agouti pigmentation) but clone 264 produced high percentage male chimeras. These chimeras were bred with C57BL/6J females and agouti offspring were generated, indicative of germline transmission of the ES cell genome.
  • Screening for the targeted mu gene was carried out by Southern blot analysis of Bgll digested DNA from tail biopsies (as described above for analysis of ES cell DNA). Approximately 50% of the agouti offspring showed a hybridizing Bgll band of 7.7 kb in addition to the wild type band of 15.7 kb, demonstrating a germline transmission of the targeted mu gene.
  • Table 1 shows the levels of serum IgM, detected by ELISA, for mice carrying both the CMD and JHD mutations (CMD/JHD), for mice heterozygous for the JHD mutation (+/JHD), for wild type (129Sv x C57BL/6J)F1 mice (+/+), and for B cell deficient mice homozygous for the JHD mutation (JHD/JHD).
  • the HCO12 human heavy transgene was generated by coinjection of the 80 kb insert of ⁇ HC2 (Taylor et al, 1994, hit. Immunol., 6: 579-591) and the 25 kb insert of pVx6.
  • the plasmid pVx6 was constructed as described below.
  • An 8.5 kb HindHI/Sall DNA fragment, comprising the germline human V HI -18 (DP- 14) gene together with approximately 2.5 kb of 5' flanking, and 5 kb of 3' flanking genomic sequence was subcloned into the plasmid vector pSP72 (Promega, Madison, WI) to generate the plasmid p343.7.16.
  • a 7 kb BamHI/Hind ⁇ i DNA fragment comprising the germline human V HS -51 (DP-73) gene together with approximately 5 kb of 5' flanking and 1 kb of 3' flanking genomic sequence, was cloned into the pBR322 based plasmid cloning vector pGP If (Taylor et al. 1992, Nucleic Acids Res. 20: 6287-6295), to generate the plasmid p251f.
  • a new cloning vector derived from pGPlf pGPlk was digested with EcoRV/BamHI, and ligated to a 10 kb EcoRV/BamHI DNA fragment, comprising the germline human V H3 -23 (DP47) gene together with approximately 4 kb of 5' flanking and 5 kb of 3' flanking genomic sequence.
  • the resulting plasmid, pi 12.2RR.7 was digested with BamHI/Sall and ligated with the 7 kb purified BamHI Sall insert of p251f.
  • the resulting plasmid, pVx4, was digested with Xhol and ligated with the 8.5 kb Xhol/Sall insert of p343.7.16.
  • a clone was obtained with the VH I -18 gene in the same orientation as the other two V genes.
  • This clone, designated pVx6, was then digested with Notl and the purified 26 kb insert coinjected with the purified 80 kb Notl insert of pHC2 at a 1 : 1 molar ratio into the pronuclei of one-half day (C57BL/6J x DBA/2 J)F2 embryos as described by Hogan et al. (B.
  • mice Hogan et al, Manipulating the Mouse Embryo, A Laboratory Manual, 2 nd edition, 1994, Cold Spring Harbor Laboratory Press, Plainview NY).
  • Three independent lines of transgenic mice comprising sequences from both Vx6 and HC2 were established from mice that developed from the injected embryos. These lines are designated (HCO12)14881, (HCO12)15083, and (HCO12)15087.
  • Each of the three lines were then bred with mice comprising the CMD mutation described in Example 1, the JKD mutation (Chen et al. 1993, EMBO J. 12: 811-820), and the (KCo5)9272 transgene (Fishwild et al. 1996, Nature Biotechnology 14: 845-851).
  • the resulting mice express human immunoglobulin heavy and kappa light chain transgenes in a background homozygous for disruption of the endogenous mouse heavy and kappa light chain loci.
  • Transgenic HuMAb Mouse® strain HC2/KCo7, having four distinct genetic modifications was used for immunizations.
  • These transgenic mice contain a human immunoglobulin gene miniloci that encodes unreananged human heavy ( ⁇ and ⁇ ) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous ⁇ and K chain loci. Accordingly, the mice exhibit no expression of mouse IgM or K, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgG ⁇ monoclonal antibodies. Mice were housed in filter cages and were evaluated to be in good physical condition on the dates of immunization, bleeds, and the day of the fusion.
  • mice that developed anti- PA titers against the protective antigen were given an i.v. injection of recombinant protective antigen seventy-two hours prior to fusion.
  • the titers from individual mice varied between 1 :200 and greater than 1 : 100,000.
  • Mouse splenocytes were harvested, purified and fused. Single-cell suspensions of splenic lymphocytes from immunized animals were fused with the murine myelanoma cell line P3X63Ag8.653 (American Type Culture Collection, Rockville MD; ATCC CRL 1580, lot F-15183) in the presence of polyethylene glycol.
  • the original ATCC vial was thawed and expanded in culture. A seed stock of frozen vials was prepared from this expansion.
  • P388D1 ATCC TIB-63 FL
  • P388D1 ATCC TIB-63 FL
  • the supernatant was spun down and filtered and used as a media addition known as conditioned media.
  • This cell line was passed for 3 to 6 months and then a new vial was thawed.
  • High Glucose DMEM Mediatech, Cellgro #10013245
  • Penicillin-Strepatientomycin Cellgro #30004030
  • High Glucose DMEM Mediatech, Cellgro #10013245
  • Penicillin-Strepatientomycin Cellgro #30004030
  • Hybridoma growth media which included: 3% Origen-Hybridoma Cloning Factor (Igen, 36335), 10% P388D1 conditioned media (8/10/99 DH), 10% FBS (Hyclone, SH30071 lot #AGH6843), L-glutamine (Gibco #1016483) 0.1% gentamycin (Gibco #1020070), 2-mercapatienthanol (Gibco #1019091) HAT (Sigma, H0262) 1.0 xlO 4 M Hypoxanthine, 4.0 xlO "7 M Aminopatienterin, 1.6 xlO "5 M Thymidine).
  • Hybridomas were selected by the addition of HAT 24 hours after fusion. Hybridomas were first screened by a sandwich ELISA for human IgG producers. Briefly, hybridoma supernatants were captured with goat anti-human kappa (Southern Biotech, Birmingham AL), and then reacted with alkaline phophatase-conjugated goat anti-human IgG (gamma-chain specific) (Jackson ImmunoResearch Laboratories, Jnc.,West Grove, PA).
  • Hybridomas producing specific human IgG-producing hybridomas were then screened using an in vitro toxin neutralization assay (Little et al., 1990, Infection and Immunity 65:5171-5175; see Example 3). Initially, the mAbs were tested for binding to anthrax protective antigen by ELISA using full length PA83. However, several mAbs that demonstrated good toxin neutralization activity (TNA) bound poorly by ELISA. Most of these antibodies reacted well with the cleaved PA63. As a result of this finding, the TNA was used as an initial screen followed by further characterization using ELISA.
  • V H and V L regions of selected anti-PA HuMabs were then isolated from RNA from hybridomas, reverse transcribed to cDNA, and V regions amplified by PCR.
  • the PCR product was sequenced as shown in Example 4.
  • HuMabs were then purified by protein A column chromatography using the following procedure: (1) Loading conditions: Supernatant was loaded on a 5 ml Protein-A column that was equilibrated with Phosphate buffered Saline (PBS); (2) Wash: PBS buffer; (3) Elution: 0.1 M Glycine with 150 mM NaCl, pH 2.9.
  • the elute was neutralized with IM Tris buffer (30ul for every 2 ml fraction). Each eluted fraction was run on gel before being pooled. Once the purity by coomassie staining was verified, fractions were pooled and dialyzed against lOmM sodium phosphate buffer with 150mM NaCl 2 , pH 7.2.
  • Example 3A In vitro neutralization of lethal toxin by anti-PA human monoclonal antibodies
  • TNA experiments were performed according to Little et al, 1990, supra. Briefly, the toxin-sensitive murine macrophage cell line J774A.1 was exposed to a mixture of PA and LF (10:1) in the presence or absence of varying concentrations of anti-PA mAbs. After a 3 hour incubation, survival of the macrophages was determined using a viability dye (MTT).
  • MTT viability dye
  • Fig. 10 demonstrate the activity of the human anti-PA antibodies to neutralize lethal toxin in vitro.
  • Table 2 indicates the effective dose of antibody required to maintain 50% viability (ED 50 ) of macrophages in the TNA.
  • ELISA plates were coated with PA83 or PA63.
  • the mAb samples were added at appropriate dilutions, and bound antibody was detected with anti-human IgG alkaline phophatase-conjugated probes.
  • the plate was developed with a p-NPP substrate and the OD 405 determined on a Softmax plate reader.
  • Binding rate constants were determined using plasmon resonance technology on a BIAcore instrument. Preliminary binding data revealed that the dissociation constants for the HuMabs are in the nanomolar range. The binding activity results (Fig.
  • HuMabs 5D5, 2H4 and 5E8 exhibited greater reactivity to PA63 than PA83, possibly because after cleavage of the antigen more binding epitopes are exposed.
  • HuMAb 5E8 To determine the activity requirement for antibody neutralization of anthrax toxin, an additional toxin neutralization assay was performed as described using HuMAb 5E8 and murine 14B7, except that the antibodies were added either before addition of toxin (-30 min.), at the same time as the addition of toxin (0 min.), 15 minutes after addition of toxin (+15 min.) or 30 minutes after the addition of the toxin (+30 min) (See Fig. 12).
  • HuMab 5E8 protects cells from anthrax toxin after exposure of cells to the toxin.
  • HuMAb 5E8 continued to prevent cell death from anthrax toxin, even when added 15 minutes post addition of the PA83 and LF.
  • HuMAb 5E8 which preferentially binds to cell-associated PA63 may be effective at rescuing cells from anthrax toxin death even after exposure. Therefore, HuMAb 5E8 and those with similar properties would be expected to have greater therapeutic activity, than antibodies that block receptor binding.
  • Example 3B In Vitro Neutralization By Humab 5E8 Requires Fc Receptor Binding A toxin neutralization assay was performed as previously described, except using the F(ab') 2 fragment of the 5E8 antibody and the intact 5E8 mAb in the absence or presence of anti-mouse FCR ⁇ / ⁇ I monoclonal antibody 2.4G2, which specifically blocks Fc receptor and antibody Fc interactions (BD Biosciences).
  • the F(ab') 2 fragment was prepared by pepsin digestion of the mAb 5E8 followed by purification of the F(ab') 2 using Protein A and Protein L column chromatography.
  • the results show that HuMab 5E8 requires Fc receptor binding for activity (see Fig. 13).
  • 2.4G2 antibody which specifically blocks Fc receptor and antibody Fc interactions, greatly reduced the antitoxin activity of huMab 5E8.
  • Example 4 Antibody Sequencing The VH and V L regions of four HuMabs were isolated from hybridoma RNA, reverse transcribed to cDNA, amplified by PCR and sequenced. The nucleic and amino acid sequences of the V H and V L regions of these HuMabs are provided below and in Figures 1-9. It is noted that the 5E8 hybridoma produces antibodies having a heavy chain that pairs with one of two light chains (V L m aj o r or V L mi nor )- Both antibodies (i.e., 5E8 VH/VL ajor and 5E8' H /VL minor) bind to anthrax PA and can neutralize anthrax toxin according to the TNA.
  • human germline sequences which produce the heavy chains of these antibodies include V H3 - 33 and V H3-7 , while human germline sequence which produce the light chains include A27, LI 8, and LI 5, as noted in Figures 1-9.
  • Example 5A ELISA Assay to Determine HuMAb 5E8 Binding Characteristics on Anthrax Protective Antigen
  • HuMAb 5E8 would not have been selected for further development if ELISA binding to PA83 was used as a primary screen, which methodology has been used previously for screening antibodies against protective antigen (see Little et al., 1988, Infection and Immunity, 56:1807-1813).
  • Example 5B ELISA Assay to Determine HuMAb 5E8 Binding Characteristics on Anthrax Protective Antigen
  • a standard ELISA was performed as described, in section Characterization of Binding of Human Monoclonal Antibodies to Protective Antigen, supra.
  • Human antibody 5E8 was incubated together with each of murine antibody 14B7, 2D5 and 1G3 (Little et al., 1996, Microbiology 142:707-715) on a plate coated with PA 63. Binding of the mouse antibody to the PA is detected with a goat anti-mouse IgG (Fc specific) alkaline phosphatase conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA).
  • Example 6 Pharmacokinetics of Human IgG after Intravenous and Subcutaneous Administration to Rabbits This Example evaluates the pharmacokinetics of human IgG after intravenous and subcutaneous administration of human monoclonal antibodies in rabbits.
  • the data were generated to support the selection of a dosing regimen for studies to evaluate the efficacy of a human monoclonal antibody against anthrax protective antigen in a lethal inhalation anthrax model in the rabbit.
  • Human IgG was administered either intravenously (2 rabbits) or subcutaneously (4 rabbits) to determine the pharmacokinetics of human monoclonal antibodies in rabbits.
  • An intravenous dose of 10 mg/kg was administered to 2 rabbits and a subcutaneous dose of 10 mg/kg was administered to 4 rabbits.
  • the Cmax (maximum concentrations) was 389 ⁇ g/mL and 155 ⁇ g/mL after intravenous and subcutaneous administration, respectively.
  • the Tmax time to maximum concentration
  • the Tmax after subcutaneous administration was 48 hours.
  • Serum concentrations greater than 100 ⁇ g/mL were observed 4 days after both intravenous and subcutaneous administration and levels greater than 20 ⁇ g/mL wereas observed after 14 days.
  • Adequate serum levels were achieved after both subcutaneous and intravenous administrations, and either dosing route is appropriate in efficacy studies. To maintain such adequate serum levels over prolonged periods, a loading dose of antibody can be administered initially, followed by a lower second dose of the antibody can be admimstered approximately 4 days after the initial dose.
  • the increased time to maximal concentration should be taken into consideration when performing subcutaneous injections in a therapeutic model.
  • Example 7A Efficacy and therapeutic treatment of human monoclonal antibodies against a lethal aerosol challenge of Bacillus anthracis (ames strain) in a rabbit inhalation model
  • This Example demonstrates the efficacy of anti-PA antibodies of the invention in a lethal inhalation anthrax model in the rabbit. Twenty-six out of 30 rabbits treated with antibody 5D5 or 5E8 survived after 14 days post-exposure to anthrax. Thus, antibodies of the invention provide an effective new treatment for exposure to anthrax.
  • Ten New Zealand White Rabbits in four groups were administered one of two monoclonal anti-PA antibodies (PA mAb 5E8 or 5D5) immediately following and 96 hours post aerosol challenge with Bacillus anthracis. Aerosolized spores were delivered via muzzle only inhalation at a dose of approximately 100 times LD50.
  • Example 7B Efficacy of the human monoclonal antibody (5E8) against a lethal aerosol challenge of Bacillus anthracis (ames strain) in a rabbit inhalation model
  • This Example demonstrates the efficacy of lower dose levels of anti-PA antibodies of the invention in a lethal inhalation anthrax model in the rabbit. Seventeen out of 20 rabbits freated with antibody 5E8 survived after 14 days post-exposure to anthrax. Thus, antibodies of the invention provide an effective treatment for exposure to anthrax.
  • Ten New Zealand White Rabbits in two groups were administered monoclonal anti-PA antibody (PA mAb 5E8) immediately following and 96 hours post aerosol challenge with Bacillus anthracis. Aerosolized spores were delivered via muzzle only inhalation at a dose of approximately 100 times LD50. Animals were observed for mortality during a 14 day post exposure period.
  • Protection by the antibody against anthrax infection is defined as an increased time-to-death or as a two week survival post challenge.
  • Table 4 below provides an evaluation of the efficacy of the human monoclonal antibodies, including number of rabbits in the study groups, dosage level of antibody admimstered 1 hour after exposure, dosage level of antibody 96 hours after exposure, and the number of rabbits surviving the study.
  • Example 7C Therapeutic treatment of the human monoclonal antibody (5E8) against a lethal aerosol challenge of Bacillus anthracis (ames strain) in a rabbit inhalation model
  • This Example demonstrates the efficacy of anti-PA antibodies of the invention in preventing mortality after clinical signs of exposure have initiated in a lethal inhalation anthrax model in the rabbit.
  • antibodies of the invention provide an effective treatment for exposure to anthrax.

Abstract

L'invention concerne des anticorps monoclonaux humains isolés se fixant sur un antigène protecteur de l'anthrax. Ces anticorps humains peuvent être produits dans un animal transgénique non humain, une souris transgénique par exemple, capable de produire des isotypes multiples d'anticorps monoclonaux humains lorsqu'il est soumis à une recombinaison VDJ et à une commutation isotypique. L'invention concerne également des dérivés d'anticorps humains (des anticorps bispécifiques et des immunoconjugués, par exemple), des compositions pharmaceutiques contenant lesdits anticorps humains, des animaux transgéniques non humains et des hybridomes produisant ces anticorps, ainsi que des méthodes thérapeutiques et diagnostiques utilisant lesdits anticorps humains.
PCT/US2004/016213 2003-05-21 2004-05-21 Anticorps monoclonaux humains diriges contre l'antigene protecteur de bacillus anthracis WO2005023177A2 (fr)

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MXPA05012571A MXPA05012571A (es) 2003-05-21 2004-05-21 Anticuerpos monoclonales humanos contra el antigeno protector de bacillus anthracis.
JP2006514938A JP4999158B2 (ja) 2003-05-21 2004-05-21 炭疽菌（ｂａｃｈｉｌｌｕｓａｎｔｈｒａｃｉｓ）の感染防御抗原に対するヒトモノクローナル抗体
AU2004270103A AU2004270103B2 (en) 2003-05-21 2004-05-21 Human monoclonal antibodies against Bacillusanthracis protective antigen
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WO2007007086A2 (fr) * 2005-07-13 2007-01-18 The Secretary Of State For Defence Anticorps contre l'anthrax
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JP2013227339A (ja) * 2006-10-02 2013-11-07 Medarex Llc Ｃｘｃｒ４に結合するヒト抗体およびその使用
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EP2161285A1 (fr) 2008-09-05 2010-03-10 Her Majesty the Queen in Right of Canada as represented by the Minister of National Defence Anticorps monoclonaux pour neutraliser la toxine d'anthrax
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JP4999158B2 (ja) 2012-08-15
US7456264B2 (en) 2008-11-25
WO2005023177A3 (fr) 2005-06-09
AU2004270103A1 (en) 2005-03-17
AU2004270103B2 (en) 2012-02-23
MXPA05012571A (es) 2006-02-08
CA2526398C (fr) 2014-07-15
EP1633785A2 (fr) 2006-03-15
EP1633785B1 (fr) 2012-11-28
JP2007525191A (ja) 2007-09-06
CA2526398A1 (fr) 2005-03-17
US20050287149A1 (en) 2005-12-29
US20120114662A1 (en) 2012-05-10
US8404820B2 (en) 2013-03-26
EP1633785A4 (fr) 2007-02-14

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